Composition for sequence determination

• US 20070172864A1
• Filed: 12/29/2006
• Published: 07/26/2007
• Est. Priority Date: 07/07/2000
• Status: Abandoned Application

• Current Assignee: James Briggs, Richard Willson, Shiao-Chun Tu, Xiaolian Gao
• Original Assignee: James Briggs, Richard Willson, Shiao-Chun Tu, Xiaolian Gao
• Inventors: Willson, Richard, Briggs, James, Tu, Shiao-Chun, Gao, Xiaolian
• Application Number:
  US11/648,164
• Publication Number:
  US 20070172864A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/133 conformational analysis
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

A sequencing methodology is disclosed that allows a single DNA or RNA molecule or portion thereof to be sequenced directly and in substantially real time. The methodology involves engineering a polymerase and/or dNTPs with atomic and/or molecular tags that have a detectable property that is monitored by a detection system.

Claims

1. A composition comprising a polymerizing agent including at least one molecular and/or atomic tag located at or near the polymerizing agent or associated with the polymerizing agent, a nucleic acid template, a primer adapted to hybridize to a complementary portion of the template to form an extendable primer-template duplex, and monomers for the polymerizing agent, where a detectable property of the tag undergoes a change before, during and/or after one or a plurality of monomer incorporations.

2. The composition of claim 1, wherein the detectable property has a first value when the polymerizing agent is in a first state and a second value when the polymerase is in a second state, and where the polymerizing agent changes from the first state to the second state and back again during one monomer incorporation or a plurality of monomer incorporations.

3. The composition of claim 2, wherein the polymerizing agent is a polymerase or reverse transcriptase.

4. The composition of claim 3, wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polymerase I.

5. The composition of claim 3, wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.

35. The composition of claim 1, wherein the monomers comprise nucleotides and the monomer tags are covalently bonded to a β

, γ

or terminal phosphate group of the tagged nucleotide types.

36. The composition of claim 35, wherein the detectable property is a fluorescent property comprising a duration, an intensity and/or frequency of emitted light.

37. The composition of claim 36, wherein each monomer type includes a fluorescent tag, and each fluorescent tag is the same or different.

38. The composition of claim 35, wherein at least two monomer types include a fluorescent tag, and each fluorescent tag is the same or different.

39. The composition of claim 35, wherein at least three monomer types include a fluorescent tag, and each fluorescent tag is the same or different.

40. The composition of claim 1, further comprising:

a collisional quencher adapted to quench fluorescence of the tagged monomers in the medium.

6. (canceled)

7. A composition comprising a polymerase or reverse transcriptase including at least one molecular and/or atomic tag located at or near the polymerizing agent or associated with the polymerase or reverse transcriptase, where a detectable property has a first value when the polymerase or reverse transcriptase is in a first state and a second value when the polymerase or reverse transcriptase is in a second state during monomer incorporation, and where the polymerase or reverse transcriptase changes from the first state to the second state and back again during each monomer incorporation.

8. The composition of claim 7, wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polymerase I.

9. The composition of claim 7, wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.

10. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. (canceled)

19. (canceled)

20. (canceled)

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. (canceled)

26. (canceled)

27. (canceled)

28. (canceled)

29. (canceled)

30. (canceled)

31. (canceled)

32. (canceled)

33. (canceled)

34. (canceled)

41. A composition comprising:

a polymerizing agent including a polymerizing agent fluorescent tag at or near the polymerizing agent or associated with the polymerizing agent,

a template,

a primer, and

monomers for the polymerizing agent, where one monomer type includes a monomer fluorescent tag covalently bonded to a part of the monomer that is release during monomer incorporation,

where at least one of the fluorescent tags has a fluorescence property that undergoes a change before, during and/or after each of a sequence of tagged monomer incorporations due to an interaction between the polymerizing agent tag and the monomer tag.

42. The composition of claim 41, wherein the polymerizing agent is a polymerase or reverse transcriptase.

43. The composition of claim 42, wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polymerase I and wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.

44. The composition of claim 43, wherein the polymerase comprises Taq DNA polymerase I having a tag attached to an amino acid at a specific amino acid position of the Taq DNA polymerase I, where the amino acid position is selected from the group consisting of 513-518, 643, 647, 649 and 653-661 of SEQ. ID No. 11.

45. The composition of claim 41, wherein the fluorescence property comprises a duration, an intensity and/or frequency of emitted fluorescent light.

46. The composition of claim 41, wherein the monomer tag and the polymerizing agent form a fluorescence resonance energy transfer (FRET) pair resulting in acceptor fluorescence, which occurs when the monomer tag and the polymerizing agent tag are in close proximity before, during and/or after each tagged monomer incorporation.

47. The composition of claim 46, wherein at least two monomer types include fluorescent tags, where the tags for each monomer type are the same or different and each monomer tag form a FRET pair with the polymerizing agent tag.

48. The composition of claim 46, wherein at least three monomer types include fluorescent tags, where the tags for each monomer type are the same or different and each monomer tag form a FRET pair with the polymerizing agent tag.

49. The composition of claim 46, wherein each monomer types include fluorescent tags, where the tags for each monomer type are the same or different and each monomer tag form a FRET pair with the polymerizing agent tag.

50. The composition of claim 41, further comprising:

a collisional quencher adapted to quench the fluorescent of the tagged monomers in the medium.

51. A composition comprising:

a polymerizing agent including a polymerizing agent fluorescent tag at or near the polymerizing agent or associated with the polymerizing agent,

a template,

a primer, and

nucleotide triphosphates (dNTPs) for the polymerizing agent, where one dNTP type includes a monomer fluorescent tag covalently bonded to the β

terminal phosphate group thereof,

where at least one of the fluorescent tags has a fluorescence property that undergoes a change before, during and/or after each of a sequence of tagged dNTP incorporations due to an interaction between the polymerizing agent tag and the dNTP tag.

52. The composition of claim 51, wherein the polymerizing agent is a polymerase or reverse transcriptase.

53. The composition of claim 51, wherein the fluorescence property comprises a duration, an intensity and/or frequency of emitted fluorescent light.

54. The composition of claim 51, wherein the dNTP tag and the polymerizing agent form a fluorescence resonance energy transfer (FRET) pair resulting in acceptor fluorescence, which occurs when the dNTP tag and the polymerizing agent tag are in close proximity before, during and/or after each tagged dNTP incorporation.

55. The composition of claim 54, wherein at least two dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.

56. The composition of claim 54, wherein at least three dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.

57. The composition of claim 54, wherein each dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.

58. The composition of claim 51, further comprising:

a collisional quencher adapted to quench the fluorescent of the tagged dNPTs in the medium.

59. A composition comprising:

a polymerizing agent including a polymerizing agent fluorescent tag at or near the polymerizing agent, associated with the polymerizing agent or covalently bonded to a site on the polymerizing agent,

a template,

a primer, and

nucleotide triphosphates (dNTPs) for the polymerizing agent, where one dNTP type includes a monomer fluorescent tag covalently bonded to the γ

phosphate group thereof,

where at least one of the fluorescent tags has a fluorescence property that undergoes a change before, during and/or after each of a sequence of tagged dNTP incorporations due to an interaction between the polymerizing agent tag and the dNTP tag.

60. The composition of claim 59, wherein the polymerizing agent is a polymerase or reverse transcriptase.

61. The composition of claim 59, wherein the fluorescence property comprises a duration, an intensity and/or frequency of emitted fluorescent light.

62. The composition of claim 59, wherein the dNTP tag and the polymerizing agent form a fluorescence resonance energy transfer (FRET) pair resulting in acceptor fluorescence, which occurs when the dNTP tag and the polymerizing agent tag are in close proximity before, during and/or after each tagged dNTP incorporation.

63. The composition of claim 62, wherein at least two dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.

64. The composition of claim 62, wherein at least three dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.

65. The composition of claim 62, wherein each dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.

66. The composition of claim 59, further comprising:

a collisional quencher adapted to quench the fluorescent of the tagged dNPTs in the medium.

67. A composition comprising:

a polymerizing agent including a polymerizing agent fluorescent tag at or near the polymerizing agent, associated with the polymerizing agent or covalently bonded to a site on the polymerizing agent,

a template,

a primer, and

nucleotide triphosphates (dNTPs) for the polymerizing agent, where one dNTP type includes a monomer fluorescent tag covalently bonded to the terminal phosphate group thereof,

where at least one of the fluorescent tags has a fluorescence property that undergoes a change before, during and/or after each of a sequence of tagged dNTP incorporations due to an interaction between the polymerizing agent tag and the dNTP tag.

69. The composition of claim 67, wherein the polymerizing agent is a polymerase or reverse transcriptase.

70. The composition of claim 67, wherein the fluorescence property comprises a duration, an intensity and/or frequency of emitted fluorescent light.

71. The composition of claim 67, wherein the dNTP tag and the polymerizing agent form a fluorescence resonance energy transfer (FRET) pair resulting in acceptor fluorescence, which occurs when the dNTP tag and the polymerizing agent tag are in close proximity before, during and/or after each tagged dNTP incorporation.

72. The composition of claim 71, wherein at least two dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.

73. The composition of claim 71, wherein at least three dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.

74. The composition of claim 71, wherein each dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.

75. The composition of claim 67, further comprising:

a collisional quencher adapted to quench the fluorescent of the tagged dNPTs in the medium.

76. A composition comprising:

a polymerizing agent including a polymerizing agent fluorescent tag at or near the polymerizing agent or associated with the polymerizing agent,

a template,

a primer, and

nucleotide triphosphates (dNTPs) for the polymerizing agent, where one dNTP type includes a dNTP fluorescent tag covalently bonded to a β and

/or γ

phosphate thereof,

where at least one of the fluorescent tags has a fluorescence property that undergoes a change before, during and/or after each of a sequence of tagged dNTP incorporations due to an interaction between the polymerizing agent tag and the dNTP tag.

77. The composition of claim 76, wherein the polymerizing agent is a polymerase or reverse transcriptase.

78. The composition of claim 77, wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polymerase I and wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.

79. The composition of claim 78, wherein the polymerase comprises Taq DNA polymerase I having a tag attached to an amino acid at a specific amino acid position of the Taq DNA polymerase I, where the amino acid position is selected from the group consisting of 513-518, 643, 647, 649 and 653-661 of SEQ. ID No. 11.

80. The composition of claim 76, wherein the fluorescence property comprises a duration, an intensity and/or frequency of emitted fluorescent light.

81. The composition of claim 76, wherein the dNTP tag and the polymerizing agent form a fluorescence resonance energy transfer (FRET) pair resulting in acceptor fluorescence, which occurs when the dNTP tag and the polymerizing agent tag are in close proximity before, during and/or after each tagged dNTP incorporation.

82. The composition of claim 81, wherein at least two dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.

83. The composition of claim 81, wherein at least three dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.

84. The composition of claim 81, wherein each dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.

85. The composition of claim 76, further comprising:

a collisional quencher adapted to quench the fluorescent of the tagged dNTPs in the medium.